Movement of intracellular compartments to their correct locations at precise times is a fundamental property of all cell types. For example, pigment cells, intestinal epithelia, lymphocytes, and neurons, require that specific organelles be targeted to precise locations at the proper time. In each case, myosin V molecular motors play key roles in organelle transport. Long-range movement of organelles occurs on microtubules via kinesin and dynein motors. Notably, the late steps in transport require transfer from kinesin to a myosin V motor, followed by movement on actin to a terminal destination. Regulation of detachment of organelles from myosin V is also critical to their proper localization. Our overall goal is to uncover mechanisms whereby myosin V regulates cellular organization. Similar to specialized cell-types in higher eukaryotes, the yeast Saccharomyces cerevisiae targets organelles to specific locations at precise times. We discovered that in coordination with the cell-cycle, a portion of the yeast vacuole is targeted from the mother cell to the bud. We further discovered that this movement requires the myosin V, Myo2. Moreover, we and others dis- covered that most intracellular movement in yeast occurs solely on actin and requires Myo2. Our recent progress provides strong evidence for the hypothesis that studies of yeast Myo2 will inform our under- standing of mammalian myosin V motors. We identified a conserved binding site for Rab GTPases, and an independent site dedicated to the exocyst subunit Sec15. The Rab GTPases and exocyst are conserved proteins that are required for secretion. That each ofthe sites on Myo2 is conserved strongly suggests that what we learn about how yeast Myo2 attaches and detaches from membranes will be directly applicable to human myosin Va, Vb and Vc. We proposed to use the yeast system to determine the mechanisms that govern myosin V-based transport. Our aims are to: 1) Determine whether binding of individual Myo2 adaptor proteins are enhanced or inhibited by other Myo2 adaptor proteins. 2) Determine whether the direct interaction of Myo2 with Ypt31/Ypt32, Sec4 and Sec15 plays roles beyond the attachment of Myo2 to secretory vesicles. 3) Determine mechanisms that regulate the detachment of myosin V from cargoes. RELEVANCE (See instructions): Intracellular transport of organelles by myosin V motors is crucial to normal cellular function, and animal physiology. Defects in myosin V based transport cause selected human diseases including neurological disorders. Our overall goal is to determine the mechanisms that regulate myosin V-based transport.